M3 subtype of muscarinic acetylcholine receptor inhibits cardiac fibrosis via targeting microRNA-29b/beta-site app cleaving enzyme 1 axis

Figures

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  Figure 1. The anti-fibrotic effects of miR-29b and it participates in the role of activating M3 receptor in cardiac fibroblasts. (A) qRT-PCR was performed to detect the relative expression of miR-29b in TGF-β1-induced cardiac fibroblasts when choline and 4-DAMP stimulation (n=6). (B-G) Relative protein level of collagen I (B, n=6; E, n=6), α-SMA (C, n=6; F, n=4), and CTGF (D, n=7; G, n=6) in cardiac fibroblasts detected by western blot. ****, P<0.0001; ***, P<0.001; **, P<0.01; *, P<0.05. GAPDH was included as a control. The data are expressed as the mean ± SEM. miR-29b, microRNA-29b; TGF-β1, transforming growth factor beta 1; 4-DAMP, 4-diphenylacetoxy-N-methylpiperidine methiodide; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; NC, negative control; α-SMA, α-smooth muscle actin; CTGF, connective tissue growth factor; AMO-miR-29b, miR-29b inhibitor; M3 receptor, M3 subtype of muscarinic acetylcholine receptor; qRTPCR, quantitative real-time reverse transcription polymerase chain reaction; SEM, standard error of mean.

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  Figure 2. Inhibition of miR-29b after M3 receptor activation increases the protein level of cardiac fibrosis markers. Relative protein level of collagen I (A, n=6), α-SMA (B, n=4), and CTGF (C, n=7) in cardiac fibroblasts detected by western blot. ***, P<0.001; **, P<0.01; *, P<0.05. GAPDH was included as a control. The data are expressed as the mean ± SEM. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; TGF-β1, transforming growth factor beta 1; AMO-miR-29b, miR-29b inhibitor; NC, negative control; miR-29b, microRNA-29b; α-SMA, α-smooth muscle actin; CTGF, connective tissue growth factor; M3 receptor, M3 subtype of muscarinic acetylcholine receptor; SEM, standard error of mean.

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  Figure 3. Inhibition of miR-29b reverses the anti-fibrotic effects of activating M3 receptor and promotes the expression of collagen I and α-SMA. (A,E) Relative collagen I protein level in cardiac fibroblasts detected by immunofluorescence. Scale bar =50 µm. (B,F) Statistical results of immunofluorescence of collagen I (B, n=6; F, n=3). (C,G) Relative α-SMA protein level in cardiac fibroblasts detected by immunofluorescence. Scale bar =50 µm. (D,H) Statistical results of immunofluorescence of α-SMA (D, n=6; H, n=3). ****, P<0.0001; ***, P<0.001; **, P<0.01; *, P<0.05. The data are expressed as the mean ± SEM. TGF-β1, transforming growth factor beta 1; AMO-miR-29b, miR-29b inhibitor; miR-29b, microRNA-29b; NC, negative control; α-SMA, α-smooth muscle actin; 4-DAMP, 4-diphenylacetoxy-N-methylpiperidine methiodide; M3 receptor, M3 subtype of muscarinic acetylcholine receptor; SEM, standard error of mean.

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  Figure 4. Inhibition of miR-29b after M3 receptor activation promotes the migration ability of cardiac fibroblasts. (A) The migration rate of cardiac fibroblasts detected by scratch test. Scale bar =500 µm. (B) Statistical results about the migration rate of cardiac fibroblasts, n=4. (C) The migration ability of cardiac fibroblasts was detected by transwell (crystal violet staining). Scale bar =150 µm. (D) Statistical results about migration ability of cardiac fibroblasts, n=4. ****, P<0.0001; ***, P<0.001; **, P<0.01. The data are expressed as the mean ± SEM. TGF-β1, transforming growth factor beta 1; AMO-miR-29b, miR-29b inhibitor; miR-29b, microRNA-29b; NC, negative control; M3 receptor, M3 subtype of muscarinic acetylcholine receptor; SEM, standard error of mean.

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  Figure 5. MiR-29b participates in the anti-fibrotic effects after M3 receptor activation through targeting BACE1. (A) Screening target genes of miR-29b by online prediction websites: miRDB, miRTarBase, TargetScan, and PicTar. (B) The binding sites of miR-29b and BACE1 in different species. (C-F) Relative protein level of BACE1 in cardiac fibroblasts detected by western blot (C, n=4; D, n=4; E, n=5; F, n=4) (G) qRT-PCR assay was used to examine the expression of miR-29b in the presence of BACE1 overexpression, n=8. (H) Relative protein level of collagen I in cardiac fibroblasts detected by western blot, n=6. ***, P<0.001; **, P<0.01; *, P<0.05. The data are expressed as the mean ± SEM. Student’s t-test was used for two-group comparisons. BACE1, beta-site app cleaving enzyme 1; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; NC, negative control; TGF-β1, transforming growth factor beta 1; miR-29b, microRNA-29b; AMO-miR-29b, miR-29b inhibitor; 4-DAMP, 4-diphenylacetoxy-Nmethylpiperidine methiodide; M3 receptor, M3 subtype of muscarinic acetylcholine receptor; qRT-PCR, quantitative real-time reverse transcription polymerase chain reaction.